PROJECT SUMMARY While tremendous progress has been made to understand mechanisms of epithelial cell injury and fibroblast activation in fibrosis, significant challenges remain in fully understanding the mechanisms of epithelial? mesenchymal crosstalk in normal lung homeostasis and during injury, repair and fibrosis. As in normal lung development, lung repair requires epithelial cells and mesenchymal cells to coordinate with each other to tune cell proliferation, migration, differentiation and apoptosis. Similarly, extracellular matrix (ECM) remodeling is mediated by a myriad of pro-fibrotic and anti-fibrotic factors that are precisely orchestrated by epithelium and mesenchyme during injury, repair and resolution. Therefore, aberrant epithelial mesenchymal interactions can lead to non-healing processes in lung repair with pathological scar formation due to myofibroblast accumulation and ECM deposition, ultimately contributing to pulmonary fibrosis. The long-term goal of this proposal is to restore epithelial-mesenchymal homeostasis in pulmonary fibrosis with transcriptional regulation of critical anti-fibrotic factors. Targeting mechanisms that augment the endogenous anti-fibrotic or fibrosis resolution signals during disease progression may serve as an attractive therapeutic strategy to alleviate lung fibrosis and restore lung function. Recently, single-cell and bulk RNA sequencing identified loss of normal epithelial cell identities and gain of abnormal indeterminate states of differentiation in IPF, with CEBPA identified as a key transcriptional regulator diminished in IPF that was common to multiple independent studies. Our central hypothesis is that CEBPA deficiency in lung epithelial cells promotes lung fibroblast activation and fibrosis, that it normally increases during repair to resolve fibrosis, and that it can be therapeutically restored using non-genome editing CRISPR gene activation to promote fibrosis resolution. This hypothesis will be tested with three specific aims: First, we will evaluate the role of Cebpa signaling from epithelial cells in maintaining lung epithelial-mesenchymal homeostasis and its protective role in pulmonary fibrosis. Second, we will determine whether increasing Cebpa expression with CRISPR gene activation is able to restore epithelial-mesenchymal homeostasis and attenuate fibroblast activation and fibrosis. We will develop and optimize an AAV-mediated approach to enhance Cebpa expression using non-genome editing CRISPR activation in the lung of aged mice that display non-resolving lung fibrosis after bleomycin injury. Lastly, We will test whether the anti-fibrotic effect of epithelial Cebpa expression is mediated by BMP4 and perform RNA-seq- based gene expression analysis of sorted epithelial cells from Cebpa gain and loss of function studies to identify additional novel candidate mediators of epithelial-mesenchymal homeostasis. Taken together, the proposed research studies will reveal critical anti-fibrotic targets for therapeutic interventions aimed at restoring epithelial-mesenchymal homeostasis and alleviating pulmonary fibrosis.